Protein Profiling Using Two‐Dimensional Difference Gel Electrophoresis (2‐D DIGE)

2-D DIGE relies on pre-electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one 2-D gel. The dye-labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with gel-to-gel variation and spot matching between gels. Image analysis programs are used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable protein abundance level changes to be identified and quantified. This unit describes the 2-D DIGE procedure including sample preparation from various cell types, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.